The basis for these studies is understanding the role that glutamylation plays in the cytotoxic activity of antifolates and its relationship to cellular folylpolyglutamates. We will utilize primarily a rodent hepatoma cell line in culture. We will evaluate the activity of the enzyme that catalyzes glutamylation, folylpolyglutamate synthetase, in cells grown under a number of conditions known to alter the rate of glutamate addition in intact cells. The turnover of methotrexate polyglutamates and folylpolyglutamates and the effect that each of these pools has on the other will be examined. A new series of folates and antifolates with a fluorine in place of hydrogen in the 4 position of the glutamate moiety will be evaluated. The fluoro substitution prevents the glutamylation of methotrexate and as such can be used to evaluate the role that glutamylation plays in the activity of the drug. Using the same rationale, reduced folate coenzymes (dihydrofolate, folinic acid, 5-methyltetrahydrofolate) will be synthesized as the 4 fluoro derivative. With these probes we will examine the transport and metabolism of the fluoroanalogs, the relationship between glutamylation of folates and growth dependence, the sensitivity of glutamylated and non-glutamylated methotrexate to toxicity prevention by purines and pyrimidines, and the role of glutamylation in rescue protocols employing folinic acid. Other areas to be investigated are the characteristics of transport-based methotrexate resistance and a more detailed analysis of the cytocidal activity of methotrexate. These studies are intended to yield greater understanding about antifolates so that they may be more selectively employed as antiproliferative agents in the treatment of neoplasia.